Polydactyly-derived allogeneic chondrocyte cell-sheet transplantation with high tibial osteotomy as regenerative therapy for knee osteoarthritis

Allogeneic cell therapies are not fully effective in treating osteoarthritis of the knee (OAK). We recently reported that transplantation of autologous chondrocyte cell-sheets along with open-wedge high tibial osteotomy promoted hyaline cartilage repair in humans. Here we describe our regenerative therapy for OAK using polydactyly-derived allogeneic chondrocyte cell-sheets (PD sheets) and temperature-responsive culture inserts. Ten patients with OAK and cartilage defects categorized arthroscopically as Outerbridge grade III or IV received the therapy. Cartilage viscoelasticity and thickness were assessed before and after transplantation. Arthroscopic biopsies obtained 12 months after transplantation were analyzed histologically. Gene expression was analyzed to evaluate the PD sheets. In this small initial longitudinal series, PD sheet transplantation was effective in treating OAK, as indicated by changes in cartilage properties. Gene marker sets in PD sheets may predict outcomes after therapy and provide markers for the selection of donor cells. This combined surgery may be an ideal regenerative therapy with disease-modifying effects in OAK patients.

The cell sheets used in this study will be used in the treatment of intractable knee cartilage using Japan's cutting-edge technology. At present, we are conducting clinical research using human stem cells with autologous chondrocyte sheets. Eight patients underwent transplantation without any major adverse events. Issues with treatment using autologous chondrocyte sheets include the sacrifice of cartilage in healthy regions, the limited number of viable donor regions, and the low growth ability of the cartilage among older adults. We expect the development of a novel treatment method that uses autologous chondrocyte sheets. In this study, we will carry out knee cartilage treatment using allogeneic chondrocyte sheets, whose therapeutic effect has been confirmed in animal experiments, in patients with knee cartilage lesions. Chondrocytes isolated from surgical remains obtained from polydactyly patients will be passaged and cryopreserved as allogeneic cells. An allogeneic chondrocyte sheet will be fabricated by culturing cells, whose safety and characteristics in a cell sheet have been confirmed in advance, from the cryopreserved cells. These sheets will be transplanted into the cartilage defect regions in the knee of patients. Thereafter, the restoration of the knee cartilage will be confirmed. The safety of this novel treatment will be objectively assessed as the primary endpoint. Furthermore, its efficacy will be assessed as the secondary endpoint. It is generally believed that joint cartilage defect, when left untreated, will progress to osteoarthritis in 10 to 20 years. Although mild cartilage damage often has no subjective symptoms, and despite fibrillation on the surface of joint cartilage, early treatment is necessary for mild cartilage damage upon confirmation of the deterioration of the extracellular matrix of chondrocytes, which plays an important role in viscoelasticity and lubrication. Once cartilage damage spreads and becomes severe, it requires major invasive surgeries, such as total knee arthroplasty, which places a huge burden on the patient. At present, autologous chondrocyte transplantation combined with periosteal transplantation is performed for moderate cartilage defects. However, this treatment poses the following problems: (1) it requires the sacrifice of tissue from two healthy regions; (2) the surgery cannot be performed multiple times due to the limited number of viable regions for tissue collection; and (3) older adults have low cartilage growth ability. Therefore, there is a demand for the development of a new treatment method.
Of the main repair methods in Japan, marrow stimulation, involves shaving the defective cartilage as thinly as possible until bleeding from the bone marrow occurs.
Thereafter, the defect is repaired using fibroblastic cartilage derived from undifferentiated mesenchymal stem cells in the bone marrow by creating multiple small holes in the bone at 3-mm to 4-mm intervals using a pointed instrument. Although the surgery could be easily performed with arthroscopy, it is best suited for individuals who are young (35 to 40 years old), have been injured within 1 year, have a lesion smaller than 2 cm 2 , and have no surgical history. Osteochondral autograft, which has become the standard method for treatment of localized osteochondral defects, involves multiple cylindrical osteochondral pillars taken from non-weight-bearing regions in the knee joint, resulting in cartilage defects at relatively large weight-bearing regions. The site of the cartilage defect is repaired by a complex transplanted autologous hyaline cartilage and the fibroblastic cartilage regenerated from the gap. Therefore, whether or not the transplanted hyaline cartilage and the regenerated fibroblastic cartilage can be bonded to each other affects the surgical results. Hence, the method is suitable for osteochondritis dissecans, including osteochondritis dissecans of the elbow and ankle joints, patella cartilage defects, cartilage defects associated with ligament and meniscus injuries, osteonecrosis, and cartilage damage secondary to trauma. Furthermore, cartilage defects should ideally be 1 cm 2 to 4 cm 2 and the patient must be 40 years of age or younger. JACC is an insurance-covered processed cellular tissue product that uses healthy periosteum and cartilage. In this method, isolated chondrocytes are embedded and cultured in atelocollagen, the defect is covered with the periosteum, and cultured cells are transplanted into the gap. However, this method is indicated for traumatic cartilage defects measuring 4 cm 2 or larger, and not for osteoarthritis (Table 1).  A report will be submitted every year, 90 days after a given period, starting from the date when the regenerative medicine provision plan is submitted to the director of the Regional Welfare Bureau.
・Notification of changes or termination of the regenerative medicine provision plan In the event that a plan for provision of regenerative medicine, etc., is to be changed (excluding minor changes), the Minister of Health, Labour and Welfare shall be notified through the director of the Regional Bureau of Health and Welfare after requesting the comments of the certified committee for regenerative medicine on the regenerative medicine provision plan, etc., after the change. The specially certified committee for regenerative medicine will be notified of minor changes within 10 days of the change.
When terminating the provision of regenerative medicine, etc., the specially certified committee for regenerative medicine shall be notified, and the notification of terminating the provision of regenerative medicine, etc., shall be submitted to the Minister of Health, Labour and Welfare through the director of the Regional Bureau of Health and Welfare within 10 days from the date of termination.

3) Method of obtaining informed consent from subjects
Prior to the start of this clinical study, the principal investigator or clinical study investigator will explain the following items to subjects based on explanatory documents and obtain written informed consent. The investigator will explain that consent is at the discretion of the subject and that they will not suffer any unfavorable treatment should they refuse consent. The name of the explaining physician, the signature of the subject, date of consent, etc., will be present in the consent form. The explanatory document and consent form will be given to the subject, and a copy will be kept in an electronic medical record.
The following items shall be described in the explanatory document. The principal investigator shall promptly inform the subject should there be information that may influence the will of subjects regarding the continuation of their participation in the clinical study. Thereafter, the principal investigator shall confirm whether the subject still wishes to continue participating in the clinical study.
Furthermore, this process will be described in the medical record.

5) Handling of personal information and protection of privacy
Since cell sheets are fabricated for each case, all physicians in the department of orthopedic surgery, including the research representative and investigators, know the subjects. Furthermore, since all clinical data are stored in electronic medical records, patient information can be accessed by physicians working at Tokai University Hospital.
Therefore, anonymization specific to this clinical study will not be performed. However, anonymity at the same level as regular inpatients will be maintained, so personal information will be managed using patient IDs.
All clinical study-related records, such as source materials, may be directly viewed during monitoring and audits of the clinical study, and investigations by the certified committee for regenerative medicine and regulatory authorities.
Furthermore, when analyzing clinical data or presenting at academic conferences, the principal investigator will assign and use patient IDs and non-regular subject identification numbers to protect personal information and ensure subject privacy.
Anonymized information, such as the correspondence Patients who meet all of the following inclusion criteria and obtain consent from a family member, who is a legally acceptable representative, will be included in the study.
① Patients of any sex from 0 to 5 years of age ② Patients with polydactyly

Exclusion criteria
Patients who meet at least one of the following exclusion criteria will be excluded from this study.
① Informed consent of a family member, who is a legal representative, cannot be obtained ② Patients with significant complications ③ Patients with an infectious disease that may pose a problem (including HBV, HCV, HIV, HTLV, and syphilis). [Rationale] [Items set in consideration of the safety of subjects].
Considering the safe and ethical performance of this clinical study and taking into account the reliable data, the items ① to ③ were set.

Discontinuation criteria
The clinical study shall be discontinued immediately after informed consent is obtained if any of the following is applicable. Considering the safe and ethical performance of this clinical study and taking into account the reliable data, the items ① to ④ were set.

Discontinuation criteria
The clinical study (cell sheet transplantation) should be discontinued immediately after informed consent is obtained, if any of the following is applicable. However, follow-up and collection of efficacy and safety data should be continued as much as possible.
[ To prevent cross-contamination, separate clean benches and incubators will be used for each sample, and only one sample will be handled at a time. Therefore, the number of cases was determined in consideration of certain restrictions on the use of the cell processing center. It took 1 to 2 years to collect, store, evaluate and validate cells for transplantation, followed by the clinical study (transplant to patients) with a 1-year follow-up period.

Enrollment
Candidacy to selection method (enrollment procedure)

1) Tissue donor
Candidacy to selection method Patients who meet the inclusion criteria will be considered candidates, and after confirming their eligibility based on medical history etc., those who have provided written informed consent will be enrolled as tissue donors.

2) Subjects (patients for transplantation)
Candidacy to selection method Those who meet the selection criteria ① and ② will be selected as candidates, and those who have submitted written consent (first time) after confirming their eligibility based on medical history will be registered as cases. However, cell sheet transplantation will be performed only on those who have submitted written consent (second time) and who meet the selection criteria ③ and ④ based on preoperative arthroscopic findings. is a chip of fresh cartilage tissue from young individuals, is being used clinically (Zimmer's DeNovo® NT). By using allogeneic chondrocytes, we can solve the issues concerning the use of the above-mentioned autologous cells. Furthermore, we believe that sufficient safety assessment can be performed by securing a sufficient number of cells with high cellular activity and assigning lot numbers. Since we will use cells isolated from tissues collected during surgery in the same facility, traceability will not be an issue.
We applied for a human stem cell clinical study because we believe that the use of a completely safe allogeneic cell sheet will result in a less invasive treatment and will have the same effect as an autologous cell sheet. With safety as the primary endpoint of this study, the efficacy of the treatment will be objectively assessed as a secondary endpoint, and various clinical evaluations will be performed to collect data on the treatment's effects.

2) Study design
As a treatment for cartilage defects in osteoarthritis, a single-arm open-label, uncontrolled, and comparative study will be conducted to confirm the safety and efficacy of cartilage repair and restoration effects of allogeneic cell sheet transplantation.

3) Methods to minimize bias
This study is designed as an exploratory study with safety assessment as the primary endpoint, and it is a single-arm open-label, uncontrolled trial.
In this study, the age and BMI of the subjects, range of cartilage defect, defect region, number of cell sheet transplants, etc., are likely important factors in efficacy assessment and will be recorded as transplant patient summary in the case report form (transplantation).
To avoid bias toward efficacy evaluation, multiple physicians will evaluate the endpoints.

4) Method
This clinical study will use chondrocytes isolated from the cartilage tissue of the knuckles to be discarded during surgery in patients who do not have infectious diseases or serious complications other than polydactyly. Most eligible patients for tissue donation are approximately a year old, so sufficient time must be given to the family, which will act as the representative, to submit informed consent; samples will be collected after obtaining the consent form. We will select cells that passed the endotoxin test, mycoplasma test, virus test, and sterility test and select cells suitable for cell karyotype analysis, cell morphology observation, and stability test. The subjects will provide informed consent twice in total: prior to preoperative arthroscopy and prior to cell sheet transplantation. The clinical study will be carried out upon obtaining each of the consent forms. The degree of the cartilage lesion during arthroscopy will be evaluated. Thereafter, we will confirm if the subject meets the selection criteria. Once the target patient has been enrolled, cell sheet preparation will begin approximately 3 weeks before the transplant date. Polydactyly-derived tissues are transported from the operating room to the cell processing center, and the cells will be isolated and cryopreserved in the same room to evaluate the properties and safety of the cells. Once the target patient has been enrolled, cell sheet preparation will begin approximately 3 weeks before the date of transplantation. The cells will be seeded in a temperature-responsive culture dish to fabricate an allogeneic chondrocyte sheet. The fabricated allogeneic chondrocyte sheet (final product) will be transplanted to the region of cartilage injury in the patient on the planned day of transplantation surgery.
Multiple sheets may be transplanted according to the size of the region of cartilage injury. If the region of cartilage injury is filled with unhealthy tissue, the tissue will be excised, the lesion will be washed, marrow stimulation will be performed, and a cell sheet will be transplanted directly above the injured region. The allogeneic chondrocyte sheet will not be sutured to the surrounding tissue.

5) Study observation period and test items <Hospitalization period>
Subjects will be hospitalized for 1 month, and postoperative rehabilitation and rest will be similar to those of subjects undergoing HTO alone.
-Content of rehabilitation- The patient's knee will be fixed with a plaster splint immediately after surgery until two weeks after surgery, and range of motion training will not be possible during this period.
Weight bearing: ・Complete non weight bearing immediately after surgery and up to 2 weeks ・One-third weight bearing from 3 weeks after partial weight bearing to 4 weeks after surgery ・One-half weight bearing from 4 to 5 weeks after surgery  Compliance tests for quality and safety will be conducted, and cells confirmed to be compatible in all items will be cryopreserved for clinical research. The patient to undergo transplantation will be enrolled, and the frozen cell will be thawed and seeded from approximately 3 weeks before the date of transplantation. After the cells reach confluence, they will be seeded to a temperature-responsive culture dish, and the allogeneic chondrocyte sheet will be prepared.
② Storage methods of specified cell products, etc.  Among the adverse events, the occurrence and incidence of serious adverse events will be confirmed, and the safety of treatment will be evaluated.
 Secondary endpoint <Efficacy assessment> 1) Clinical assessment: Tegner-Lysholm Knee Scoring Scale, Japanese Knee Injury and Osteoarthritis Outcome Score assessments will be performed preoperatively and at 1 month, 3 months, 6 months, and 1 year after surgery as a clinical evaluation criterion.
2) X-rays: These will be used to assess the joint space, subchondral bone condition, and the progression of arthropathy. The progression of arthropathy will be objectively assessed using Kellgren-Lawrence grading scale before surgery and at 1 month, 3 months, 6 months, and 1 year after surgery.

3) MRI:
Changes in cartilage thickness and properties over time will be objectively assessed using Nelson MRI Grading preoperatively, and 1 month, 3 months, 6 months, and 1 year postoperatively.

4) Arthroscopy:
This will be used to evaluate the cartilage properties (color, hardness, and smoothness) and Outerbridge classification of the joint surface 1 year after surgery. If any injury or swelling of the joint occurs, the test shall be performed as appropriate to evaluate the condition of the cartilage.

5) Photoacoustic evaluation:
To quantitatively evaluate the viscoelastic properties of joint cartilage 1 year after surgery, we will evaluate the cartilage in the transplant region and surrounding cartilage under arthroscopy using an in-house functional diagnostic device. This is a functional evaluation method that has already been clinically applied at Tokai University Hospital under the approval of the Clinical Study Review Board of Tokai University School of Medicine.

6) Histological evaluation:
A part of the regenerated tissue will be submitted for biopsy at the time of arthroscopy, Safranin-O staining will be performed, and an objective histological evaluation will be performed using the Modified Mankin Score. [Endpoints] ① Scores in clinical evaluation criteria at 1 year after surgery Regarding the subjects' data, information will be collected from the principal investigator and the investigator in charge of the subjects. The collaborators will compile a case report form, confirm it with the data manager, and then report it to the investigator. Data on safety and effectiveness will be reported regularly and to the Tokai University certified committee for regenerative medicine in accordance with the Regenerative Medicine Safety Assurance Act. . Therefore, we believe that there is an extremely low possibility that the allogeneic chondrocyte sheet will be immunorejected. Furthermore, the safety of the allogeneic cell sheet will be confirmed by tumorigenicity test, stability test, and karyotype analysis. Moreover, various virus tests will be conducted multiple times to ensure sufficient safety. We believe that the risk of adverse events is at the same level as that of regular knee surgery under general anesthesia, and it is unlikely that their occurrence will be specific to this study.

Handling of adverse events 1) Symptoms or diseases
Any unfavorable or unintended sign, symptom, or disease occurring after surgery will be treated as an adverse event. If a complication worsens, it will also be handled as an adverse event. If the severity of the efficacy endpoint worsens, the event will not be treated as an adverse event.
2) Objective opinion Regardless of the items specified or not specified in this study protocol, the adverse events during onset, during maximal deterioration, and during outcome determination, as well as the data necessary for determining association will be stated in the medical record.

3) Recording and investigation of adverse events
In this clinical study, after the provision of regenerative medicine and so on as a general rule, regular outpatient visits and tests (such as clinical evaluation, X-ray, MRI examination, and arthroscopy) for 5 years or more will be carried out in outpatient clinics, similar to postoperative follow-up of ordinary surgical therapy. In addition to verifying the effects of this regenerative medicine and so on we will confirm the presence or absence of diseases and so on caused by the provision of this regenerative medicine and so on and grasp and follow up the occurrence of diseases, etc.
If an adverse event occurs, its symptoms or disease, content of objective findings, date of onset, degree, severity, presence or absence of treatment and its content, outcome and its determination date, and relevance to this clinical study with reasons will be stated in the medical records. If the name of the disease is stated, the symptoms associated with the disease will not be noted as an adverse event.
If adverse events are observed in the symptoms or diseases observed during the treatment period or objective findings, regardless of whether there is a causal relationship with this clinical study, in principle, follow-up will be conducted until the condition stabilizes or recovers to a level that cannot be considered as an adverse event.
However, this does not apply if the investigator confirms that the patient has recovered.
In that case, the grounds for determining recovery shall be stated in the medical record.
If irreversible adverse events are observed due to organic disorders (including cerebral infarction and myocardial infarction), follow-up will be conducted until the symptoms stabilize or resolve.

4) Classification of adverse events
The severity of adverse events will be classified according to the following criteria:

6) Serious adverse events
If serious adverse events occur during the treatment period, whether associated with the clinical study or not, the principal investigator or the investigators will take appropriate measures immediately for the subject. The investigator will promptly report to the head of the hospital. In response to the report, the implementation regulations will be followed. In addition, even if this clinical study has fewer than 10 cases, the study will be discontinued. In the event of a disease, disability, death, or infectious disease suspected to be due to the provision of regenerative medicine, the principal investigator shall promptly report to the hospital director.
The providing institution manager will report diseases and so on to the certified committee for regenerative medicine and the Minister of Health, Labour and Welfare, in accordance with Ministerial Ordinance No. 110 (Articles 35 and 36). Furthermore, in the event of a situation that may have a significant impact on ensuring the safety of the specified processed cell product, the product manufacturer shall report the serious situation in accordance with Article 107 of the Ministerial Ordinance.

8) Measures to understand information
The presence of diseases and so on will be confirmed in the outpatient clinic, and information such as health status will be grasped. When a disease and so on suspected to be caused by the provision of this regenerative medicine and so on occurs, a certain follow-up period will be provided to grasp information on the disease, etc.
We will establish a system that allows us to obtain the contact information of those who have received this regenerative medicine and so on and to grasp the outbreak of illness, etc. Necessary information will be grasped from the records such as the electronic medical record system of the hospital.

9) Providing new information
If new information on the safety of this clinical study is obtained, the practitioner will promptly submit a written report to the hospital director, dean of the school of medicine, principal investigator and investigators of the clinical study. The principal investigator and investigators will provide additional explanation to the subject and revise the explanatory document and informed consent form as necessary.

Medical fees and compensation 1) Medical fees
All costs related to cell sheet transplantation are paid from the research funds of Tokai University School of Medicine, with no out-of-pocket payments from the patient.

2) Compensation
If the clinical study results in compensation or liability for health damage to subjects, insurance for clinical studies, such as regenerative medicine, and compensation is possible within the scope of the compensation.

Statistical considerations 1) Target enrollments and rationale
Based on the results of clinical studies conducted to date, we estimated a target response rate of 75% and a threshold response rate of 25%. Assuming that the result exceeds the threshold response rate, the required number of cases calculated with α=0.05 and 1-β (detection power)=0.9 is eight cases. Considering the feasibility of the study, 10 cases were set.

2) Analysis set
Cell sheet transplantation is performed.

3) Analysis items and methods
① Analysis items: The secondary endpoint, the Japanese Knee Injury and Osteoarthritis Outcome Sco re (J-KOOS) score 1 year after surgery, will be evaluated from the perspective of i mproving clinical symptoms.
② Analysis methods: The amount of change in J-KOOS (value 1 year after surgery -preoperative valu e) in the target population will be analyzed by one-way analysis of variance and multiple comparison test (post hoc Holm-Bonferroni).
<Handling of missing data or dropouts> If there are missing data 1 year after surgery and dropouts less than 1 year after surgery (evaluation points), the final J-KOOS score from the scores obtained 1 month, 3 months, and 6 months after surgery will be used for evaluation.

4) Interim analysis
Since the target enrollment is as small as 10 and the endpoint is 1 year after surgery, no interim analysis is planned. However, for cases with poor improvement of JKOOS as an efficacy evaluation, the assessment will be carried out each time.

5) Changes in the original statistical analysis plan
In the event of changes in the original statistical analysis plan, the protocol and statistical analysis plan will be revised, and the changes will be explained in the summary report. An application for changes to the certified committee for regenerative medicine will be submitted, and after receiving the opinion of the committee, the Minister of Health, Labour and Welfare will be notified via the director of the regional welfare bureau. The results obtained in this study may be published in academic conferences, papers, etc. At the time of presentation, the subject's confidentiality (privacy) will be preserved.
Furthermore, evaluation and analysis will be performed with the endpoint at one year of the observation period. A summary report will be submitted to the Ministry of Health, Labour and Welfare, and will be published on jRCT.

Use of samples and information in new research
The samples and information obtained in this study are extremely valuable and can only be obtained through surgery. Specimens and collected data that could not be used as surplus or for transplantation may be anonymized and used for other research for the development of medicine with the consent of the subject or the surrogate (see consent form). It may also be discarded without being used for transplantation.
16. Management of samples and information (storage and disposal) 1) Sample storage, storage period, and disposal Samples, such as a part of cells, and a part of a specific processed cell product will be stored for 10 years by consignment to a storage room or reagent adjustment room affiliated with the cell processing facility, cell therapy archive, etc. After the storage period, the samples will be processed to prevent identification of the individual and then disposed of as medical waste in accordance with waste management regulations.
2) Information management, storage period, and disposal The handling, management, storage, and storage period of the records related to the test are as follows.
Records related to regenerative medicine, etc.: Electronic data will be managed and stored in electronic medical records and hard disks dedicated to clinical research, and paper-based original materials, case report forms, and materials related to tests will be stored in lockers with keys. The documents will be stored for 30 years in accordance with the Act on Securing Safety of Regenerative Medicine.
17. Study funding and conflicts of interest of investigators, etc.
This research will be carried out with research funding from the Japan Agency for Medical Research and Development. In addition, the research representative has received research funding from CellSeed Co., Ltd., and is conducting joint research such as optimizing the cell sheet manufacturing method and improving the culture equipment. However, since the funds provided will not be used for this clinical study, the transparency of the study implementation and the reliability of the results will not be compromised. In addition, the research representative of this clinical study will report to the Conflict-of-Interest Management Committee of the University and undergo review. The Institutional Review Board (certified committee for regenerative medicine) has recognized the appropriateness of the implementation of this study. 19. Response to the provision of medical care to subjects after study As a general rule, follow-up will be carried out for at least 5 years in outpatient clinics, as in the case of postoperative follow-up of regular surgery.

Monitoring and audit
Monitoring will be carried out by people in charge designated by the principal investigator from among the investigators and collaborators in this research. As for the implementation procedure, the test implementation procedure will be confirmed, documents will be viewed, and the storage status will be confirmed by on-site monitoring according to the procedure manual. After confirmation of the monitoring results from the monitoring manager, a report will be submitted to the principal investigator. When an audit of medical institutions and so on is conducted, the principal investigator shall cooperate in the source document verification, etc.